The global demand for fructooligosaccharides (FOS) is growing due to human health benefits associated with their consumption. FOS are prebiotics that selectively stimulate the growth of bifidobacteria, thereby promoting colonic health [1,2]. Further claims as to the effect of FOS consumption relate to mineral absorption, lipid metabolism and the control of type II diabetes and have been extensively reviewed [2-4]. Further to their health benefits, FOS are used in the food industry as low calorie sweeteners. They are also added to food products to improve their organoleptic properties and their inclusion allows producers to label their products as ‘functional foods’—a claim that resonates with health conscious consumers [2,3].
It is well known that some β-fructofuranosidases possess the ability to transform sucrose to FOS. β-fructofuranosidases are family 32 glycoside hydrolase (GH32) enzymes that act on sucrose and related β-D-fructofuranosides [5]. They are also known as invertases (EC 3.2.1.26) as they hydrolyse sucrose to produce invert sugar—an equimolar mixture of dextrorotatory D-glucose and levorotatory D-fructose [6]. Crystal structures for GH32 β-fructofuranosidases reveal that the enzymes display a bimodular arrangement of a N-terminal catalytic domain containing a five-bladed β-propeller fold linked to a C-terminal β-sandwich domain [7-10]. β-fructofuranosidases hydrolyse β-glycosidic bonds by a double displacement catalytic mechanism that retains the configuration of the fructose anomeric carbon [11]. Multiple sequence alignments (MSAs) identified a highly conserved aspartate close to the N terminus that serves as the catalytic nucleophile and a glutamate residue that acts as a general acid/base catalyst [12]. The β-fructofuranosidases which are capable of transforming sucrose to FOS possess fructosyltransferase activity whereby the sugar moiety is transferred from the enzyme-fructosyl intermediate to an acceptor other than water [7,13]. This reaction forms the basis of FOS synthesis from sucrose. Enzymes from Aspergillus spp. [14-16] and Aureobasidium pullulans [17] exhibit good propensities for the synthesis of inulin type FOS from sucrose, with β-(2→1) linkages between fructose units.
Synthesis of FOS (GFn) from sucrose (GF) occurs via a disproportionation reaction with the reaction generalised as GFn+GFn→GFn−1+GFn+1, [18,19]. In a batch reaction the initial products are glucose and 1-kestose (GF2), and as the reaction progresses, nystose (GF3) and β-fructofuranosyl nystose (GF4) levels increase. Reaction conditions influence the dominance of hydrolytic or transferase reactions with high substrate concentrations favouring the latter [14].
Industrial biotransformation of sucrose to FOS is currently conducted in a batch system using the β-fructofuranosidase from A. niger ATCC 20611 (subsequently classified as A. japonicus). The enzyme is added to a buffered 50-60% (wt/vol) sucrose solution with the reaction proceeding at 50-60° C. for up to 20 hours [19]. These severe industrial conditions impose limitations on activity. The fructosyltransferase activity of the enzyme has been shown to be non-competitively inhibited by the glucose product, limiting complete sucrose conversion [19]. Furthermore, long-term enzyme stability is severely compromised at temperatures above 50° C. despite immobilisation efforts [20].
There is thus still a need for alternative enzymes which are able to efficiently convert sucrose to FOS on an industrial scale.